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Metalloprotease from Chryseobacterium

Active Publication Date: 2017-04-27
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to an isolated polypeptide with protease activity, which can be used in detergent compositions and in cleaning processes such as laundry and hard surface cleaning. The isolated polypeptide has at least 60% sequence identity to a metalloprotease from Chryseobacterium sp. The invention also includes polynucleotides encoding the polypeptides, constructs, expression vectors, and host cells comprising these polynucleotides. The isolated polypeptide has at least 60% sequence identity to a metalloprotease from Chryseobacterium sp. The invention also includes a method for producing the polypeptide by culturing a host cell comprising the polynucleotide. The isolated polypeptide has at least 60% sequence identity to a metalloprotease from Bacillus clausii and at least 60% sequence identity to a metalloprotease from Bacillus amyloliquefaciens.

Problems solved by technology

Metalloproteases are a group of proteases which have not yet been commercially applied in detergent industry, mainly due to low stability in detergent compositions as well as under the conditions during the wash process.
However, known metalloproteases are very unstable under conventional wash conditions and in conventional detergent compositions.
Thus, the use of metalloproteases in detergents and in wash and cleaning processes has not yet been commercialized.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

nd Expression of a M4 Metalloprotease from Chryseobacterium sp. 10696

[0353]The metalloprotease was derived from a bacterial strain received as NCIMB1314; originally isolated from a sample of minced fish meat and identified as a Flavobacterium sp. Chromosomal DNA from a pure culture was purified and subjected to full genome sequencing using Illumina technology. The assembled genome sequence and subsequent analysis of the 16S ribosomal subunit gene sequences indicated that the correct taxonomical identification was as a new species within the genus Chryseobacterium and the strain was named Chryseobacterium sp. 10696.

[0354]The genome sequence was analyzed for metalloproteases from the MEROPS family M4 by comparison to the M4 protease NprE from B. subtilis 168 (Uniprot P68736) by search using the BLAST program. This analysis identified a gene encoding a putative M4 metalloprotease with the nucleotide sequence given in SEQ ID: 1.

[0355]The gene encoding the M4 metalloprotease was amplifie...

example 2

ion of the M4 Protease from Chryseobacterium sp. 10696

[0360]The supernatant was filtered through a Nalgene 0.2 μm filtration unit in order to remove the rest of the Bacillus host cells. Solid (NH4)2SO4 was added to the 0.2 μm filtrate to a final concentration of 1.5M (NH4)2SO4 and the M4 protease solution was applied to a PhenylToyopearl column (from TosoHaas) equilibrated in 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, 1.5M (NH4)2SO4, pH 6. After washing the column extensively with the equilibration buffer, the M4 protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, pH 6. The eluted peak was applied to a Bacitracin agarose column (from Upfront chromatography) equilibrated in 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, pH 6. After washing the column extensively with the equilibration buffer, the M4 protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, 1M NaCl, pH 6 with 25% (v / v) 2-propanol. The eluted peak was transferred to 20 mM MES, 2 mM CaCl2, pH 6 on a G25 sephadex column (f...

example 3

ization of the Chryseobacterium Protease

[0363]The Protazyme OL characterization assay was used for obtaining the pH-activity profile at 37° C., the pH-stability profile (residual activity after 2 hours at indicated pH-values) and the temperature-activity profile at pH 7. For the pH-stability profile the protease was diluted 7× in the different characterization assay buffers to reach the pH-values of these buffers and incubated for 2 hours at 37° C. After incubation, the pH of the protease incubations was transferred to pH 7, before assay for residual activity, by dilution in the pH 7 assay buffer.

[0364]The results are shown in Tables 6-8 below. Data for Neutrase (M4 protease from Bacillus amyloliquefaciens) are included in the tables. For Table 6, the activities are relative to the optimal pH for the enzymes. For Table 7, the activities are residual activities relative to a sample, which was kept at stable conditions (5° C., pH 7). For Table 8, the activities are relative to the opt...

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Abstract

The present invention relates to a new metalloprotease and the use thereof in cleaning processes, such as laundry and dish wash. The invention also relates to detergent compositions and cleaning compositions comprising a metalloprotease.

Description

REFERENCE TO SEQUENCE LISTING[0001]This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to cleaning and / or detergent compositions comprising metalloproteases (E.C 3.4.24). The invention further concerns new metalloproteases from Chryseobacterium sp. and the use of thereof in cleaning processes, such as dish wash and laundry. Further the invention concerns methods of doing cleaning, such as dish wash and laundry.BACKGROUND OF THE INVENTION[0003]The detergent industry has for more than 30 years implemented different enzymes in detergent formulations, most commonly used enzymes includes proteases, amylases and lipases each adapted for removing various types of stains. In addition to the enzymes detergent compositions typically include a complex combination of ingredients. For example, most cleaning products include surfactant system, bleaching agen...

Claims

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Application Information

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IPC IPC(8): C11D3/386C12N9/52C11D11/00
CPCC11D3/386C12N9/52C11D11/0017C11D2111/12
Inventor GJERMANSEN, MORTENNYMAND-GRARUP, SOERENOESTERGAARD, PETER RAHBEKREISER, ANNA VERENABJERRE, JEANNETTE
Owner NOVOZYMES AS
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